bioseparation -protin

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Chapter 1

Protein Bioseparation: An Overview

1.1 Introduction

Protein bioseparation which refers to the recovery and purification of pro-

tein products from various biological feed streams is an important unit

operation in the food, pharmaceutical and biotechnological industry. For

the purpose of simplicity, these industries will be collectively referred to

as bioprocess industriesthroughout this book. Protein bioseparation is at

the present moment more important in the bioprocess industry than at any

time before. This is largely due to the phenomenal developments in re-

cent years in the field of modern biotechnology. More and more protein

products have to be purified in larger quantities. A further boost to protein

bioseparation is likely to come from the developing science ofproteomics.

The purpose of this chapter is to provide the reader with an overview

on protein bioseparation. Different aspects of protein bioseparation are

discussed in the book edited by Sadana [1]. In order to read aboutbiosep-

arationsin general, refer to the book by Belteret al. [2].

1.2 Proteins

A protein is a biopolymer composed of basic building blocks called aminoacids. Naturally occurring proteins are made up of up to 20 different amino

acids. Proteins are by far the most abundant biopolymers in living cells

(constituting about 40 to 70 percent of dry cell weight) and have diverse

biological functions:

a. Structural components (e.g. collagen, keratin)

b. Catalysts (e.g. enzymes, catalytic antibodies)

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2 Protein Bioseparation Using Ultrafiltration

c. Transport molecules (e.g. haemoglobin, serum albumin)

d. Regulatory substances (e.g. hormones)e. Protective molecules (e.g. antibodies)

A protein molecule can be a single poly-(amino acid) chain or may

comprise more than one poly-(amino acid) chain, held together by cova-

lent bonds or by non-covalent interactions. A protein usually coils up and

folds into a specific 3-dimensional configuration, depending on the intrin-

sic properties of the protein as well as on the environment in which theprotein exists. The structure of a protein can be defined at different levels,

these being:

a. Primary

b. Secondary

c. Tertiary

d. Quaternary

The primary structure of a protein is the sequence of amino acids

present in the poly-(amino acid) chain/s. The secondary structure describes

the local structure of linear segments of the protein molecule. The three

most common types of secondary structure are the alpha helices, the beta

sheets, and the turns. The tertiary structure is the three-dimensional ar-

rangement of all the atoms present in a single poly-(amino acid) chain. The

quaternary structure describes the arrangement of the poly-(amino acid)

chains (or subunits) in a particular protein. For details on proteins, refer to

the following books [35].

1.3 Protein products

As mentioned in the previous section, proteins have a diverse range ofbiological functions. Proteins also have a diverse array of applications. A

large number of protein based products have been commercialised. These

can be classified into the following broad categories:

a. Food and nutritional products

b. Pharmaceutical products

c. Industrial catalysts


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Protein Bioseparation: An Overview 3

d. Diagnostic products

e. Proteins used for other miscellaneous applications

Some of the protein-based products are listed in Table 1.1. The first

two named categories follow intuitively from the importance of proteins in

living systems. A large number of protein products are used as foods, food

additives and as nutraceuticals. These are obtained from various micro-

bial, plant and animal sources. Depending on their specific applications,

these need to be processed (e.g. purified) to varying degrees. By the rule of

thumb, nutraceuticals have greater purity requirements than do food addi-

tives and these in turn have to be processed to a greater extent than foods.

Pharmaceutically useful proteins are frequently referred to as thera-

peutic proteins. Most of the recent developments in the area of protein

bioseparation are centred on therapeutic proteins.

Enzymes, which are biological catalysts, can be usedin vitrofor indus-trial scale catalysis. These enzymes are referred to as industrial enzymes

and are produced in large quantities. Another major use of enzymes is in

diagnostics. Enzymes are also used as components of detergent formula-

tions and cosmetic products.

1.4 The requirement for protein bioseparation

Most protein-based products need to be purified before they can be used.

The need for purification is due to the following:

a. Reduction in bulk

b. Concentration enrichment

c. Removal of specific impurities (e.g. toxins from therapeutic products)

d. Prevention of catalysis other than the type desired (as with enzymes)

e. Prevention of catalysis poisoning (as with enzymes)

f. Recommended product specifications (e.g. pharmacopoeial

requirement)

g. Enhancement of protein stability

h. Reduction of protein degradation (e.g. by proteolysis)


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Table 1.1. Protein products.

Proteins

Food / Food additives / Nutraceuticals

Egg albumin

Casein

Soy proteins

Whey protein concentrate

Protein hydrolysates

Alpha lactalbumin

Beta lactoglobulin

Lysozyme

Pharmaceuticals

Monoclonal antibodies

Serum albumin

Serum immunoglobulins

Factor VIII

Tissue plasminogen activator

Urokinase

Streptokinase

Insulin

Erythropoietin

Alpha and beta interferon

Factor IX

Industrial enzymes

Hemmicellulase

Glucose isomerase

Alpha amylase

Penicillin G acylase

Alkaline proteases

Cellulases

Diagnostic enzymes

Peroxidase

Glucose oxidase

Miscellaneous

Enzymes used in cosmetic products

Detergent enzymes

Digestive enzymes

Enzyme based silage additive


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Some of the characteristic features of most protein products are:

a. These products are present at very low concentrations in their respective

biological feed streams

b. These products, are present, along with large numbers of impurities,

some of which are only slightly different from the products themselves

c. These products are thermolabile

d. These products are sensitive to operating conditions, such as pH and

salt concentratione. These products are sensitive to chemical substances, such as surfactants

and solvents

f. The quality requirements for these products are frequently very

demanding

These above-mentioned factors imply that an ideal protein biosepara-

tion process must combine high productivity with high selectivity of sepa-

ration, and must be feasible atmildoperating conditions.

1.5 Economic aspects of protein bioseparation

The isolation and purification of proteins from the product streams of

bioreactors and other biological feed streams is widely recognised to be

technically and economically challenging. Protein bioseparation quite of-ten becomes the limiting factor in the successful development of protein

based products. The isolation and purification cost can be a substantial

fraction of the total cost of production for most products of biological ori-

gin. Table 1.2 shows the bioseparation cost as approximate proportion of

cost of production for certain protein based products. As clearly indicated

by these figures, bioseparation cost is the major cost and this is an incentive

for developing cost-effective isolation and purification processes.

1.6 Protein bioseparation methods

A myriad of protein bioseparation techniques is available. Some of these

protein isolation and purification techniques are discussed in the following

books [1, 610]. When developing a bioseparation process for a specific


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Table 1.2. Cost of protein bioseparation.

Product Approximate relative Bioseparation cost as % of total

price cost of production

Food/additives 1 1030

Nutraceuticals 210 3050

Industrial enzymes 510 3050

Diagnostic enzymes 50100 5070

Therapeutic enzymes 50500 6080

protein, the following should be taken into consideration:

a. The volume or flow rate of the feed stream

b. The relative abundance of the protein in the feed stream

c. A profile of the impurities present

d. The intended application of the protein, along with particular product

specificationse. The market price of the protein

Protein bioseparation techniques can be classified into three broad

categories:

a. High-productivity, low-resolution

b. High-resolution, low-productivity

c. High-resolution, high-productivity

Most conventional protein bioseparation processes rely on a scheme,

which is best described as RIPP (Removal, Isolation, Purification and

Polishing) [2]. Biological feed streams are generally dilute with respect

to the target proteins, which need to be separated from a large number of

impurities. Such a feed stream would easily overwhelm a high-resolution

separation device. Therefore, low-resolution, high-productivity techniquesare used first to reduce the volume and the overall concentration of the pro-

cess stream. This is followed by high-resolution, low-productivity tech-

niques to obtain the pure target protein. However, with the advent of

high-resolution, high-productivity techniques, it is frequently possible to

shorten, if not totally replace the RIPP scheme.

Table 1.3 lists some of the more commonly used protein bioseparation

techniques. Note that ultrafiltration is listed in two categories since the


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Table 1.3. Protein bioseparation techniques.

High-productivity,low-resolution

Cell disruption

Precipitation

Centrifugation

Liquid-liquid extraction

Microfiltration

Ultrafiltration

Supercritical fluid extraction

High-resolution,low-productivity

Ultracentrifugation

Packed bed chromatography

Affinity separation

Electrophoresis

Supercritical fluid chromatography

High-resolution, high-productivity

Fluidised bed chromatography

Membrane chromatography

Ultrafiltration

Monolith column chromatography

resolution in an ultrafiltration process depends very much on how it is

operated. Some of the other protein bioseparation techniques are brieflydiscussed below.

1.6.1 Cell disruption

Different types of cells (e.g. microbial, animal and plant) produce pro-

teins either intracellularly or extracellularly. For recovering intracellular

proteins, the cells have to be disrupted. Different cell disruption techniquesare listed in Table 1.4.

1.6.2 Precipitation

Proteins can be partially purified using precipitation techniques. The

main advantage of these techniques is that very large process volumes

can be handled. Proteins can be precipitated using (a) salting out salts


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Table 1.4. Cell disruption methods.

Physical methods

Disruption in ball mill or pebble mill

Disruption using colloid mill

Disruption using French press

Disruption using ultrasonic vibrations

Chemical methods

Disruption using detergents

Disruption using enzymesCombination of detergent and enzymes

Disruption using solvents

like ammonium sulfate and sodium chloride, (b) solvents like ethanol,

methanol and acetone, and (c) concentrated acids or alkali. Precipitation

processes are generally favoured at low temperatures. After precipitation,

the precipitates are separated from the bulk liquid (also called the super-

natant) using centrifugation or filtration.

1.6.3 Centrifugation

A centrifuge is a device that is used for separating precipitated proteins

from a solution by spinning the samples at rotation speeds typically rang-

ing from 100010000 revolutions per minute. Centrifugation may be car-ried out at two different scales:

a. Analytical centrifugation

b. Preparative centrifugation

Analytical centrifuges are used in research laboratories and in the in-

dustry for small-scale separation and sample preparations (i.e., 11000ml). Preparative centrifuges handle larger sample volumes (i.e., 1 to sev-

eral thousand litres).

1.6.4 Ultracentrifugation

An ultracentrifuge is a special type of centrifuge, which is operated at a

much higher speed, e.g. 30000 revolutions per minute. Ultracentrifuges


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of both analytical and preparative scales are available. These are used to

separate proteins in solution.

1.6.5 Column chromatography

Chromatography relies on the distribution of components to be separated

between two phases: a stationary (or binding) phase and a mobile phase,

which carries these components through the stationary phase. In its sim-

plest form the stationary phase is present in the form of a packed bed withina column, hence the term column chromatography. The mixture of com-

ponents enters a chromatographic column along with the mobile phase,

and each individual component is flushed through the system at a different

rate. The rate of migration of a component depends on its interactions with

the stationary phase, as well as on the mobile phase flow rate. Different

types of columns are used for chromatographic separations. Packed beds

are most commonly used. Other types include packed capillary columns,open tubular columns and monolith columns.

Different types of separation chemistries are used for chromatographic

separation of proteins:

a. Ion exchange

b. Reverse phase partitioning

c. Hydrophobic interaction

d. Size exclusion

e. Supercritical fluid extraction

f. Affinity interaction

1.6.6 Electrophoresis

Electrophoresis refers to the separation of components by employing their

electrophoretic mobility (i.e., movement in an electric field). The mixture

is added to a conductive medium, followed by the application of an electric

field across it. Positively charged components will migrate towards the

negative electrode, negatively charged components will migrate towards

the positive electrode, while neutral components will remain immobile.

Electrophoresis can be classified into two types, depending on the medium


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in which the separation is carried out:

a. Gel electrophoresis

b. Liquid phase electrophoresis

1.6.7 Membrane chromatography

Column chromatography has several major limitations. Some of these lim-

itations could be overcome by using synthetic microporous membranes as

chromatographic media [11,12]. In membrane chromatography, the trans-

port of proteins to their binding sites takes place by convection and hence

these processes are fast. Thus, the high resolution of a chromatographic

process can be combined with the high productivity of a membrane sepa-

ration process.

1.6.8 Microfiltration

Microfiltration relies on the use of microporous membranes for the sepa-

ration of micron-sized particles from fluids. The various applications of

microfiltration include:

a. Cell harvesting from bioreactors

b. Virus removal for solutions

c. Clarification of fruit juice and beveragesd. Water purification

e. Air filtration (for sterilisation)

f. Media sterilisation in bioreactors

References

1. A. Sadana (ed.), Bioseparation of Proteins Academic Press, New York

(1998).

2. P.A. Belter, C.L. Cussler and W.-S. Hu,Bioseparations John Wiley and Sons,

New York (1988).

3. T.E. Creighton, Proteins, 2nd Edition W.H. Freeman and Company, New

York (1993).

4. T.E. Creighton (ed.), Protein Folding W.H. Freeman and Company, New York

(1992).


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5. A. Fersht, Structure and Mechanism in Protein Science W.H. Freeman and

Company, New York (1999).

6. S. Roe (ed.),Protein Purification Techniques, 2nd EditionOxford University

Press, Oxford (2001).

7. R.K. Scopes,Protein Purification: Principles and PracticeSpringer-Verlag,

New York (1982).

8. F. Franks (ed.),Protein BiotechnologyHumana Press, New Jersey (1993).

9. G. Walsh and D.R. Headon, Protein Biotechnology John Wiley and Sons,

Chichester (1994).

10. S. Doonan (ed.),Protein Purification Protocols Humana Press, New Jersey

(1996).

11. K.G. Briefs and M.R. Kula, Fast protein chromatography on analytical and

preparative scale using modified microporous membranes Chemical Engi-

neering Science47 (1992) 141.

12. D.K. Roper and E.N. Lightfoot, Separation of biomolecules using adsorptive

membranesJournal of Chromatography A 702 (1995) 3.

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